Engineering a Pseudo-26-kDa Schistosoma Glutathione Transferase from bovis/haematobium for Structure, Kinetics, and Ligandin Studies

Glutathione transferases (GSTs) are the main detoxification enzymes in schistosomes. These parasitic enzymes tend to be upregulated during drug treatment, with Schistosoma haematobium being one of the species that mainly affect humans. There is a lack of complete sequence information on the closely related bovis and haematobium 26-kDa GST isoforms in any database. Consequently, we engineered a pseudo-26-kDa S. bovis/haematobium GST (Sbh26GST) to understand structure–function relations and ligandin activity towards selected potential ligands. Sbh26GST was overexpressed in Escherichia coli as an MBP-fusion protein, purified to homogeneity and catalyzed 1-chloro-2,4-dinitrobenzene-glutathione (CDNB-GSH) conjugation activity, with a specific activity of 13 μmol/min/mg. This activity decreased by ~95% in the presence of bromosulfophthalein (BSP), which showed an IC50 of 27 µM. Additionally, enzyme kinetics revealed that BSP acts as a non-competitive inhibitor relative to GSH. Spectroscopic studies affirmed that Sbh26GST adopts the canonical GST structure, which is predominantly α-helical. Further extrinsic 8-anilino-1-naphthalenesulfonate (ANS) spectroscopy illustrated that BSP, praziquantel (PZQ), and artemisinin (ART) might preferentially bind at the dimer interface or in proximity to the hydrophobic substrate-binding site of the enzyme. The Sbh26GST-BSP interaction is both enthalpically and entropically driven, with a stoichiometry of one BSP molecule per Sbh26GST dimer. Enzyme stability appeared enhanced in the presence of BSP and GSH. Induced fit ligand docking affirmed the spectroscopic, thermodynamic, and molecular modelling results. In conclusion, BSP is a potent inhibitor of Sbh26GST and could potentially be rationalized as a treatment for schistosomiasis.

Phase transitions in natural C-O-H-N-S fluid inclusions - implications for gas mixtures and the behavior of solid H2S at low temperatures

C–O–H–N–S-bearing fluids are known as one of the most challenging geochemical systems due to scarcity of available experimental data. H2S-rich fluid systems were recognized in a wide array of world-class mineral deposits and hydrocarbon reservoirs. Here we report on a nature of low-temperature (T ≥ −192 °C) phase transitions observed in natural CH4–H2S–CO2–N2–H2O fluid inclusions, which are modeled as closed thermodynamic systems and thus serve as natural micro-laboratories representative of the C–O–H–N–S system. For the first time, we document solid–solid H2S (α ↔ β ↔ γ) transitions, complex clathrates and structural transformations of solid state H2S in natural inclusion gas mixtures. The new data on Raman spectroscopic features and a complete sequence of phase transition temperatures in the gas mixtures contribute to scientific advancements in fluid geochemistry. Enhanced understanding of the phase equilibria in the C–O–H–N–S system is a prerequisite for conscientious estimation of P-T-V-X properties, necessary to model the geologic evolution of hydrocarbon and mineral systems. Our findings are a driver for the future research expeditions to extraterrestrial H2S-rich planetary systems owing to their low temperature environments.

Experience in genetic testing of hypertrophic cardiomyopathy using nanopore DNA sequencing

Aim. To investigate the application of the Oxford Nanopore Technologies’ third generation sequencing for the genetic testing of hypertrophic cardiomyopathy.Material and methods. The study involved 12 patients with hypertrophic cardiomyopathy aged 18 to 67 years (women, 9; men, 3). Using the PCR barcoding amplicons (SQK-LSK109) protocol, DNA libraries were created which contained long-range PCR fragments of the MYH7, MYBPC3, TNNT2, TNNI3 and TPM1 genes. The sequencing was performed using the MinION system by Oxford Nanopore Technologies (UK). Bioinformatic algorithms for data analysis included Guppy v.5.0.7, Nanopolish and Clairvoyante. The identified genetic variants were confirmed by Sanger sequencing.Results. Data on the complete sequence of the five major sarcomeric genes for hypertrophic cardiomyopathy were obtained. We found eight potentially disease-causing sequence variants in MYH7, MYBPC3 and TNNT2 genes by monomolecular sequencing. However, only three mutations p.Arg243Cys, p.Tyr609Asn, p.Arg870His in the MYH7 gene, and one mutation p.Lys985Asn in the MYBPC3 were confirmed by Sanger sequencing. Cascade screening of pathogenic variant p.Arg870His in the MYH7 gene was performed. We found one asymptomatic carrier.Conclusion. It appears that monomolecular sequencing technology is a feasible approach to identify mutations in patients with hypertrophic cardiomyopathy. Although improvement in accuracy of DNA sequencing, as well as optimization and simplification of bioinformatic algorithms for identification of the genetic variants are needed.

Contour Fitting of Fused Filaments Cross-Section Images by Lemniscates of Booth: Application to Viscous Sintering Kinetics Modeling

A method for image analysis was implemented to determine the edge pixels of two biopolymer-based thermoplastic filaments during their hot melt isothermal sintering at 120 °C. Successive inverted ellipses are adjusted to the contour of the sintered filaments and lead to the identification of the parameters of the corresponding lemniscates of Booth. The different steps of the morphological image analysis are detailed, from 8-bit coded acquired images (1 frame/s), to the final fitting of the optimized mathematical functions describing the evolution of the filaments envelope. The complete sequence is composed of an initial pure viscous sintering step during the first minute, followed by viscoelastic swelling combined with melt spreading for a longer time, and then the stabilization of the sintered filaments shape for over 2 min at high temperatures. Using a master curve obtained from Hopper’s abacus, the characteristic viscous sintering time is assessed at tvs = 78 s, confirming the one previously found based on the measurement of the bonding neck length alone. Then, the full description of the evolution of the thermoplastic filaments envelope is assessable by image analysis during sintering trials as a result of its digital modeling as successive lemniscates of Booth, reflecting geometry changes in the molten state.

In-Depth Sequence Analysis of Bread Wheat VRN1 Genes

The VERNALIZATION1 (VRN1) gene encodes a MADS-box transcription factor and plays an important role in the cold-induced transition from the vegetative to reproductive stage. Allelic variability of VRN1 homoeologs has been associated with large differences in flowering time. The aim of this study was to investigate the genetic variability of VRN1 homoeologs (VRN-A1, VRN-B1 and VRN-D1). We performed an in-depth sequence analysis of VRN1 homoeologs in a panel of 105 winter and spring varieties of hexaploid wheat. We describe the novel allele Vrn-B1f with an 836 bp insertion within intron 1 and show its specific expression pattern associated with reduced heading time. We further provide the complete sequence of the Vrn-A1b allele, revealing a 177 bp insertion in intron 1, which is transcribed into an alternative splice variant. Copy number variation (CNV) analysis of VRN1 homoeologs showed that VRN-B1 and VRN-D1 are present in only one copy. The copy number of recessive vrn-A1 ranged from one to four, while that of dominant Vrn-A1 was one or two. Different numbers of Vrn-A1a copies in the spring cultivars Branisovicka IX/49 and Bastion did not significantly affect heading time. We also report on the deletion of secondary structures (G-quadruplex) in promoter sequences of cultivars with more vrn-A1 copies.

Unified framework for localized patterns in reaction–diffusion systems; the Gray–Scott and Gierer–Meinhardt cases

A recent study of canonical activator-inhibitor Schnakenberg-like models posed on an infinite line is extended to include models, such as Gray–Scott, with bistability of homogeneous equilibria. A homotopy is studied that takes a Schnakenberg-like glycolysis model to the Gray–Scott model. Numerical continuation is used to understand the complete sequence of transitions to two-parameter bifurcation diagrams within the localized pattern parameter regime as the homotopy parameter varies. Several distinct codimension-two bifurcations are discovered including cusp and quadruple zero points for homogeneous steady states, a degenerate heteroclinic connection and a change in connectedness of the homoclinic snaking structure. The analysis is repeated for the Gierer–Meinhardt system, which lies outside the canonical framework. Similar transitions are found under homotopy between bifurcation diagrams for the case where there is a constant feed in the active field, to it being in the inactive field. Wider implications of the results are discussed for other pattern-formation systems arising as models of natural phenomena. This article is part of the theme issue ‘Recent progress and open frontiers in Turing’s theory of morphogenesis’.

Molecular recording of sequential cellular events into DNA

Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cell's genome, allowing for the later reconstruction of cellular experiences using high-throughput DNA sequencing. Existing DNA recorders have achieved high-information recording, durable recording, prolonged recording over multiple timescales, multiplexed recording of several user-selected signals, and temporally resolved signal recording, but not all at the same time. We present a DNA recorder called peCHYRON (prime editing Cell HistorY Recording by Ordered iNsertion) that does. In peCHYRON, prime editor guide RNAs (pegRNAs) insert a variable triplet DNA sequence alongside a constant propagation sequence that deactivates the previous and activates the next step of insertion. This process results in the sequential accumulation of regularly spaced insertion mutations at a synthetic locus. Accumulated insertions are permanent throughout editing because peCHYRON uses a prime editor that avoids cutting both DNA strands, which risks deletions. Editing continues indefinitely because each insertion adds the complete sequence needed to initiate the next step. Constitutively expressed pegRNAs generate insertion patterns that support straightforward reconstruction of cell lineage relationships. Pulsed expression of different pegRNAs enables the reconstruction of pulse sequences, which may be coupled to biological stimuli for temporally-resolved multiplexed event recording.

A complete sequence of mitochondrial genome of Neolamarckia cadamba and its use for systematic analysis

Neolamarckia cadamba is an important tropical and subtropical tree for timber industry in southern China and is also a medicinal plant because of the secondary product cadambine. N. cadamba belongs to Rubiaceae family and its taxonomic relationships with other species are not fully evaluated based on genome sequences. Here, we report the complete sequences of mitochondrial genome of N. cadamba, which is 414,980 bp in length and successfully assembled in two genome circles (109,836 bp and 305,144 bp). The mtDNA harbors 83 genes in total, including 40 protein-coding genes (PCGs), 31 transfer RNA genes, 6 ribosomal RNA genes, and 6 other genes. The base composition of the whole genome is estimated as 27.26% for base A, 22.63% for C, 22.53% for G, and 27.56% for T, with the A + T content of 54.82% (54.45% in the small circle and 54.79% in the large circle). Repetitive sequences account for ~ 0.14% of the whole genome. A maximum likelihood (ML) tree based on DNA sequences of 24 PCGs supports that N. cadamba belongs to order Gentianales. A ML tree based on rps3 gene of 60 species in family Rubiaceae shows that N. cadamba is more related to Cephalanthus accidentalis and Hymenodictyon parvifolium and belongs to the Cinchonoideae subfamily. The result indicates that N. cadamba is genetically distant from the species and genera of Rubiaceae in systematic position. As the first sequence of mitochondrial genome of N. cadamba, it will provide a useful resource to investigate genetic variation and develop molecular markers for genetic breeding in the future.

A new straton in the Late Devonian sedimentation in the Tsil’ma river basin of the Middle Timan

Research subject. Terrigenous rocks of the Sargaevo stage of sedimentation in various structural-facies settings within the Tsilma area (Middle Timan). Material and methods. The composition, structural features of rocks, and the species diversity of spores of higher plants were studied in the most complete natural outcrops proposed as a stratotype of the Paladinskaya Formation – a new straton of the Sargaevo age in the Middle Timan. Over a large area of the region, the deposits contain very few remains of fossil fauna, often indifferent. As a result, palynostratigraphy was selected as the main biostratigraphic method. Results. In the Middle Timan, a new straton, the Paladinskaya Formation, is described, which characterizes the Sargaevo stage of sedimentation in various structural-facies settings within the Tsil’ma area. The new straton has clear lithological and detailed palynological characteristics; a description of the limitotypes was performed.At present, the Paladinskaya Formation is the most complete sequence of Early Frasnian sedimentation in the European North-East of Russia, where it was possible to record regional responses to the global Frasnes event and to determine the position of the controversial level of the boundary between the Middle and Upper Devonian. Conclusions. The name “Ust’e Yarega” Formation in the Middle Timan cannot be considered valid, since it is a homonym of the sediments of the same age in the South Timan. The section is represented mainly by continental, rather than marine, deposits. A new name for the identifed formation is proposed – “Paladinskaya” with a description of the stratum section and limitotype.The Paladinskaya Formation is characterized by subcomplexes of spores (А and Б) of the regional miospore zone Cristatisporites pseudodeliquescens, which made it possible to correlate the marine and continental deposits of the Early Frasnian on the territory of the entire Timan-Pechora region and establish its exact position in the stratigraphic scheme of the Devonian.

Complete genome sequence and construction of an infectious full-length cDNA clone of a cucumber vein yellowing virus (CVYV) isolate from Portugal

Cucumber vein yellowing virus (CVYV) is a member of the genus Ipomovirus in the family Potyviridae. In the National Center for Biotechnology Information (NCBI) database, three complete genome sequences of CVYV isolates from Spain (NC_006941), Israel (KT276369), and Jordan (JF460793) are available. In this study, we report the complete sequence of an isolate of CVYV from Portugal (DSMZ PV-0776) along with the construction of an infectious full-length cDNA clone via Gibson assembly. The sequence of CVYV Portugal shows the closest relationship to a CVYV isolate from Spain (genome, 99.7% identity; polyprotein, 99.7% identity). The CVYV full-length cDNA clone was introduced by electroporation into Rhizobium radiobacter and infiltrated into the cotyledons of Cucumis sativus plantlets, resulting in symptoms resembling those of the wild-type virus. Transmission of the infectious CVYV full-length clone by the whitefly Bemisia tabaci was confirmed. This first report confirming the infectivity of a CVYV cDNA clone provides the opportunity to study gene functions in a consistent genomic background.